Somatostatin agonists

ABSTRACT

Claimed are a series of somatostatin agonists typically characterized by alkylation of the amide nitrogen, and uses thereof. Examples of claimed compounds are those according to formula (I), 
 
A 1 -cyclo{Cys-A 2 -D-Trp-A 3 -A 4 -Cys}-A 5 -Y 1 ,  (I) 
wherein: 
     A 1  is an optionally substituted D- or L-aromatic α-amino acid or optionally substituted D- or L-cyclo(C 3-6 )alkylalanine;    A 2  is an optionally substituted aromatic α-amino acid or optionally substituted cyclo(C 3-6 )alkylalanine;    A 3  is Lys or Orn;    A 4  is β-Hydroxyvaline, Ser, hSer, or Thr;    A 5  is β-Hydroxyvaline, Ser, hSer, or Thr; and    Y 1  is OH, NH 2  or NHR 1 , where R 1  is (C 1-6 )alkyl; 
 
wherein each said optionally substituted aromatic α-amino acid and each said optionally substituted cyclo(C 3-6 )alkylalanine is optionally substituted with one or more substituents each independently selected from the group consisting of halogen, NO 2 , OH, CN, (C 1-6 )alkyl, (C 2-6 )alkenyl, (C 2-6 )alkynyl, (C 1-6 )alkoxy, Bzl, O-Bzl, and NR 9 R 10 , where R 9  and R 10  each is independently H or (C 1-6 ) alkyl; and 
 
wherein the amine nitrogen of each peptide bond and the amino group of A 1  of formula (I) is optionally substituted with a methyl group, provided that there is at least one said methyl group; and further provided that said compound is not D-Phe-cyclo{Cys-Phe-D-Trp-Lys-(N-Me-Thr)-Cys}-Thr-NH 2 ; 
or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of pending U.S. Ser. No.10/474,248, filed Apr. 26, 2004, which is a United States nationalfiling under 35 U.S.C. §371 of international (PCT) application No.PCT/US02/10882, filed Apr. 8, 2002, designating the United States, andclaiming priority to U.S. provisional application No. 60/282,526, filedApr. 9, 2001.

BACKGROUND OF THE INVENTION

Somatostatin (SRIF), a tetradecapeptide discovered by Brazeau et al.,has been shown to have potent inhibitory effects on various secretoryprocesses in tissues such as pituitary, pancreas and gastrointestinaltract. SRIF also acts as a neuromodulator in the central nervous system.These biological effects of SRIF, all inhibitory in nature, are elicitedthrough a series of G protein coupled receptors, of which five differentsubtypes have been characterized (SSTR-1 to SSTR-5). These five subtypeshave similar affinities for the endogenous SRIF ligands but havediffering distribution in various tissues. SRIF binds to the fivedistinct receptor (SSTR) subtypes with relatively high and equalaffinity for each subtype. SRIF produces a variety of effects, includingmodulation of hormone release, e.g., growth hormone, glucagon, insulin,amylin, and neurotransmitter release. Some of these effects have beenassociated with its binding to a specific SRIF receptor. For example,the inhibition of growth hormone has been attributed to the somatostatintype-2 receptor (“SSTR-2”) (Raynor, et al., Molecular Pharmacol. 43:838(1993); Lloyd, et al., Am. J. Physiol. 268:G102 (1995)), while theinhibition of insulin has been attributed to the somatostatin type-5receptor (“SSTR-5”) (Coy, et al. 197:366-371 (1993)). Activation oftypes 2 and 5 have been associated with growth hormone suppression andmore particularly GH secreting adenomas (acromegaly) and TSH secretingadenomas. Activation of type 2 but not type 5 has been associated withtreating prolactin secreting adenomas.

As is well known to those skilled in the art, SRIF and analogs thereofare useful in the treatment of a great variety of diseases and/orconditions. An exemplary but by no means exhaustive list of suchdiseases and/or conditions would include: Cushings Syndrome (see Clark,R. V. et al, Clin. Res. 38, p. 943A, 1990); gonadotropinoma (see AmbrosiB., et al., Acta Endocr. (Copenh.) 122, 569-576, 1990);hyperparathyroidism (see Miller, D., et al., Canad. Med. Ass. J., Vol.145, pp. 227-228, 1991); Paget's disease (see, Palmieri, G. M. A., etal., J. of Bone and Mineral Research, 7, (Suppl. 1), p. S240 (Abs. 591),1992); VIPoma (see Koberstein, B., et al., Z. Gastroenterology, 28,295-301, 1990 and Christensen, C., Acta Chir. Scand. 155, 541-543,1989); nesidioblastosis and hyperinsulinism (see Laron, Z., Israel J.Med. Sci., 26, No. 1, 1-2, 1990, Wilson, D. C., Irish J. Med. Sci., 158,No. 1, 31-32, 1989 and Micic, D., et al., Digestion, 16, Suppl. 1.70.Abs. 193, 1990); gastrinoma (see Bauer, F. E., et al., Europ. J.Pharmacol., 183, 55 1990); Zollinger-Ellison Syndrome (see Mozell, E.,et al., Surg. Gynec. Obstet., 170, 476-484, 1990); hypersecretorydiarrhea related to AIDS and other conditions (due to AIDS, see Cello,J. P., et al., Gastroenterology, 98, No. 5, Part 2, Suppl., A163 1990;due to elevated gastrin-releasing peptide, see Alhindawi, R., et al.,Can. J. Surg., 33, 139-142, 1990; secondary to intestinal graft vs. hostdisease, see Bianco J. A., et al., Transplantation, 49, 1194-1195, 1990;diarrhea associated with chemotherapy, see Petrelli, N., et al., Proc.Amer. Soc. Clin. Oncol., Vol. 10, P 138, Abstr. No. 417 1991); irritablebowel syndrome (see O'Donnell, L. J. D., et al., Aliment. Pharmacol.Therap., Vol. 4., 177-181, 1990); pancreatitis (see Tulassay, Z., etal., Gastroenterology, 98, No. 5, Part 2, Suppl., A238, 1990); Crohn'sDisease (see Fedorak, R. N., et al., Can. J. Gastroenterology, 3, No. 2,53-57, 1989); systemic sclerosis (see Soudah, H., et al.,Gastroenterology, 98, No. 5, Part 2, Suppl., A129, 1990); thyroid cancer(see Modigliani, E., et al., Ann., Endocr. (Paris), 50, 483-488, 1989);psoriasis (see Camisa, C., et al., Cleveland Clinic J. Med., 57 No. 1,71-76, 1990); hypotension (see Hoeldtke, R. D., et al., Arch. Phys. Med.Rehabil., 69, 895-898, 1988 and Kooner, J. S., et al., Brit. J. Clin.Pharmacol., 28, 735P-736P, 1989); panic attacks (see Abelson, J. L., etal., Clin. Psychopharmacol., 10, 128-132, 1990); sclerodoma (see Soudah,H., et al., Clin. Res., Vol. 39, p. 303A, 1991); small bowel obstruction(see Nott, D. M., et al., Brit. J. Surg., Vol. 77, p. A691, 1990);gastroesophageal reflux (see Branch, M. S., et al., Gastroenterology,Vol. 100, No. 5, Part 2 Suppl., p. A425, 1991); duodenogastric reflux(see Hasler, W., et al., Gastroenterology, Vol. 100, No. 5, Part 2,Suppl., p. A448, 1991); Graves' Disease (see Chang, T. C., et al., Brit.Med. J., 304, p. 158, 1992); polycystic ovary disease (see Prelevic, G.M., et al., Metabolism Clinical and Experimental, 41, Suppl. 2, pp76-79, 1992); upper gastrointestinal bleeding (see Jenkins, S. A., etal., Gut., 33, pp. 404-407, 1992 and Arrigoni, A., et al., AmericanJournal of Gastroenterology, 87, p. 1311, (abs. 275), 1992); pancreaticpseudocysts and ascites (see Hartley, J. E., et al., J. Roy. Soc. Med.,85, pp. 107-108, 1992); leukemia (see Santini, et al., 78, (Suppl. 1),p. 429A (Abs. 1708), 1991); meningioma (see Koper, J. W., et al., J.Clin. Endocr. Metab., 74, pp. 543-547, 1992); and cancer cachexia (seeBartlett, D. L., et al., Surg. Forum., 42, pp. 14-16, 1991).

Other indications associated with activation of the SRIF receptorsubtypes are inhibition of insulin and/or glucagon and more particularlydiabetes mellitus, angiopathy, proliferative retinopathy, dawnphenomenon and Nephropathy; inhibition of gastric acid secretion andmore particularly peptic ulcers, enterocutaneous andpancreaticocutaneous fistula, irritable bowel syndrome, Dumpingsyndrome, watery diarrhea syndrome, AIDS related diarrhea,chemotherapy-induced diarrhea, acute or chronic pancreatitis andgastrointestinal hormone secreting tumors; treatment of cancer such ashepatoma; inhibition of angiogenesis, treatment of inflammatorydisorders such as arthritis; retinopathy; chronic allograft rejection;angioplasty; preventing graft vessel and gastrointestinal bleeding.

It is preferred to have an analog which is selective for the specificSRIF receptor subtype or subtypes responsible for the desired biologicalresponse, thus, reducing interaction with other receptor subtypes whichcould lead to undesirable side effects. Further, because of the shorthalf-life of native SRIF, various SRIF analogs have been developed,e.g., for the treatment of acromegaly. (Raynor, et al., MolecularPharmacol. 43:838 (1993)) The development of potent, smaller SRIFagonists led to the discovery of differing affinities of the varioustruncated ligands for the different subtypes. It appears that Trp⁸-Lys⁹residue often is present in ligands that are recognized by the receptor.The Trp⁸-Lys⁹ residue forms part of a β-bend which is usually stabilizedvia substitution of D- for L-Trp, cyclization of the backbone, adisulfide bridge, or all constraints. One unintended consequence of suchstructural simplification, carried out before the discovery of multiplereceptor subtypes, was the loss of broad spectrum binding affinity. Thisis typified by the high type 2 but low type 1, 3, 4, and 5 affinities ofpeptides in the OCTREOTIDE® series. Thus, the many basic biologicalstudies with this type of analogue failed to detect effects mediated byall but one of the SRIF receptor subtypes.

We have discovered that peptide backbone constraint can be introduced byN-alkylation. This modification largely restricts the affected residueand the amino acid preceding it to an extended conformation andadditionally blocks potential intramolecular hydrogen bonding sites andalso proteolytic enzyme cleavage sites thus potentially enhancingpharmacokinetic properties of a peptide. Only a few N-methyl amino acidsare commercially available and their synthesis is tedious. However, inanother aspect of the present invention, we have discovered a procedureto N-methylate truncated SRIF analogues at every amino acid residueusing the solid-phase procedure, adopted from that reported by Millerand Scanlan, (J. Am. Chem. Soc. 1997, 119, 2301-2302).

In one aspect the invention relates to a peptide according to formula(I):A¹-cyclo{Cys-A²-D-Trp-A³-A⁴-Cys}-A⁵-Y¹,  (I)wherein:A¹ is an optionally substituted D- or L-aromatic α-amino acid oroptionally substituted D- or L-cyclo(C₃₋₆)alkylalanine;A² is an optionally substituted aromatic α-amino acid or optionallysubstituted cyclo(C₃₋₆)alkylalanine;A³ is Lys or Orn;A⁴ is β-Hydroxyvaline, Ser, hSer, or Thr;A⁵ is β-Hydroxyvaline, Ser, hSer, or Thr; andY¹ is OH, NH₂ or NHR¹, where R¹ is (C₁₋₆)alkyl;wherein each said optionally substituted aromatic α-amino acid and eachsaid optionally substituted cyclo(C₃₋₆)alkylalanine is optionallysubstituted with one or more substituents each independently selectedfrom the group consisting of halogen, NO₂, OH, CN, (C₁₋₆)alkyl,(C₂₋₆)alkenyl, (C₂₋₆)alkynyl, (C₁₋₆)alkoxy, Bzl, O-Bzl, and NR⁹R¹⁰,where R⁹ and R¹⁰ each is independently H or (C₁₋₆) alkyl; andwherein the amine nitrogen of each peptide bond and the amino group ofA¹ of formula (I) is optionally substituted with a methyl group,provided that there is at least one said methyl group; and furtherprovided that said compound is notD-Phe-cyclo{Cys-Phe-D-Trp-Lys-(N-Me-Thr)-Cys}-Thr-NH₂;or a pharmaceutically acceptable salt thereof.

A preferred group of compounds of formula (I) are those compoundswherein:

A¹ is Phe, D-Phe, Tyr, D-Tyr, β-Nal, D-β-Nal, Cha or D-Cha;

A² is Phe, Tyr, β-Nal or Cha; and

Y¹ is OH or NH₂;

or a pharmaceutically acceptable salt thereof.

A preferred group of compounds of the immediately foregoing group ofcompounds are those compounds wherein A¹ is D-Phe or Tyr; or wherein A²is Phe; or wherein A³ is Lys; or wherein A⁴ is Thr; or wherein A⁵ isThr; or a pharmaceutically acceptable salt thereof.

In a still more preferred embodiment the invention features a compoundof formula (I) wherein said compound is according to the formula:

-   (N-Me-D-Phe)-{Cys-Phe-D-Trp-Lys-Thr-Cys}-Thr-NH₂;-   D-Phe-{(N-Me-Cys)-Phe-D-Trp-Lys-Thr-Cys}-Thr-NH₂;-   D-Phe-cyclo{Cys-(N-Me-Phe)-D-Trp-Lys-Thr-Cys}-Thr-NH₂;-   D-Phe-{Cys-Phe-(N-Me-D-Trp)-Lys-Thr-Cys}-Thr-NH₂;-   D-Phe-{Cys-Phe-D-Trp-(N-Me-Lys)-Thr-Cys}-Thr-NH₂;-   D-Phe-cyclo{Cys-Phe-D-Trp-Lys-Thr-(N-Me-Cys)}-Thr-NH₂;-   D-Phe-cyclo{Cys-Phe-D-Trp-Lys-Thr-Cys}-(N-Me-Thr)-NH₂;-   (N-Me-Tyr)-{Cys-Phe-D-Trp-Lys-Thr-Cys}-Thr-NH₂;-   Tyr-{(N-Me-Cys)-Phe-D-Trp-Lys-Thr-Cys}-Thr-NH₂;-   Tyr-{Cys-(N-Me-Phe)-D-Trp-Lys-Thr-Cys}-Thr-NH₂;-   Tyr-{Cys-Phe-(N-Me-D-Trp)-Lys-Thr-Cys}-Thr-NH₂;-   Tyr-{Cys-Phe-D-Trp-(N-Me-Lys)-Thr-Cys}-Thr-NH₂;-   Tyr-{Cys-Phe-D-Trp-Lys-(N-Me-)Thr-Cys}-Thr-NH₂;-   Tyr-{Cys-Phe-D-Trp-Lys-Thr-(N-Me-Cys)}-Thr-NH₂; or-   Tyr-{Cys-Phe-D-Trp-Lys-Thr-Cys}-(N-Me-Thr)-NH₂;    or a pharmaceutically acceptable salt thereof.

In another aspect, the invention features a compound according toformula (II),

wherein:

A¹ is a D- or L- isomer of Ala, Leu, Ile, Val, Nle, Thr, Ser, β-Nal,β-Pal, Trp, Phe, 2,4-dichloro-Phe, pentafluoro-Phe, p-X-Phe, or o-X-Phe,wherein X is CH₃, Cl, Br, F, OH, OCH₃ or NO₂;

A² is Ala, Leu, Ile, Val, Nle, Phe, β-Nal, pyridyl-Ala, Trp,2,4-dichloro-Phe, pentafluoro-Phe, o-X-Phe, or p-X-Phe, wherein X isCH₃, Cl, Br, F, OH, OCH₃ or NO₂;

A³ is pyridyl-Ala, Trp, Phe, β-Nal, 2,4-dichloro-Phe, pentafluoro-Phe,o-X-Phe, or p-X-Phe, wherein X is CH₃, Cl, Br, F, OH, OCH₃ or NO₂;

A⁶ is Val, Ala, Leu, Ile, Nle, Thr, Abu, or Ser;

A⁷ is Ala, Leu, Ile, Val, Nle, Phe, β-Nal, pyridyl-Ala, Trp,2,4-dichloro-Phe, pentafluoro-Phe, o-X-Phe, or p-X-Phe, wherein X isCH₃, Cl, Br, F, OH, OCH₃ or NO₂;

A⁸ is a D- or L-isomer of Ala, Leu, Ile, Val, Nle, Thr, Ser, Phe, β-Nal,pyridyl-Ala, Trp, 2,4-dichloro-Phe, pentafluoro-Phe, p-X-Phe, oro-X-Phe, wherein X is CH₃, Cl, Br, F, OH, OCH₃ or NO₂;

each R₁ and R₂, independently, is H, lower acyl or lower alkyl; and R₃is OH or NH₂;

wherein the amine nitrogen of each of the amide peptide bonds and theN-terminal amino acid is optionally substituted with a methyl group,

provided that there is at least one said methyl group in a compound offormula (II); that at least one of A¹ and A⁸ and one of A² and A⁷ mustbe an aromatic amino acid; and that A¹, A², A⁷ and A⁸ cannot all bearomatic amino acids;

or a pharmaceutically acceptable salt thereof.

In one embodiment the invention features a compound according to formula(II) wherein said compound is selected from the list consisting of:

-   H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Thr-Phe-Thr-NH₂;-   H-D-Phe-p-NO₂-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH₂;-   H-D-Nal-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH₂;-   H-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH₂;-   H-D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH₂;-   H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH₂; and-   H-D-Phe-Ala-Tyr-D-Trp-Lys-Val-Ala-β-D-Nal-NH₂;    or a pharmaceutically acceptable salt thereof.

In another embodiment the invention features a peptide selected from thelist of peptides, denoted “group III”, consisting of:

-   D-β-Nal-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-β-Nal-NH₂;-   D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-β-Nal-NH₂;-   D-β-Nal-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH₂;-   D-Phe-Cys-Phe-D-Trp-Lys-Thr-Pen-Thr-NH₂;-   D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-OH;-   D-Phe-Cys-Phe-D-Trp-Lys-Thr-Pen-Thr-OH;-   Gly-Pen-Phe-D-Trp-Lys-Thr-Cys-Thr-OH;-   Phe-Pen-Tyr-D-Trp-Lys-Thr-Cys-Thr-OH;-   Phe-Pen-Phe-D-Trp-Lys-Thr-Pen-Thr-OH;-   H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol;-   H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   H-D-Trp-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂;-   H-D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂;-   H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH₂;-   H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂;-   Ac-D-Phe-Lys*-Tyr-D-Trp-Lys-Val-Asp-Thr-NH₂ (an amide bridge formed    between Lys* and Asp);-   Ac-hArg(Et)-2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(Et)-2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(Bu)-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(Et)-2-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-L-hArg(Et)-2-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(CH₂CF₃)₂-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(CH₂CF₃)₂-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(CH₂CF₃)₂-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Phe-NH₂;-   Ac-D-hArg(CH₂CF₃)₂-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NHEt;-   Ac-L-hArg(CH₂CF₃)₂-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(CH₂CF₃)₂-Gly-Cys-Phe-D-Trp-Lys(Me)-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(CH₂CF₃)₂-Gly-Cys-Phe-D-Trp-Lys(Me)-Thr-Cys-Thr-NH Et;-   Ac-hArg(CH₃; hexyl)-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   H-hArg(hexyl₂)-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(Et)-2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NHEt;-   Ac-D-hArg(Et)-2-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Phe-NH₂;-   Propionyl-D-hArg(Et)-2-Gly-Cys-Phe-D-Trp-Lys(iPr)-Thr-Cys-Thr-NH₂;-   Ac-D-β-Nal-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Gly-hArg(Et)₂-NH₂;-   Ac-D-Lys(iPr)-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(CH₂CF₃)₂-D-hArg(CH₂CF₃)₂-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-D-hArg(CH₂CF₃)₂-D-hArg(CH₂CF₃)₂-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Phe-NH₂;-   Ac-D-hArg(Et)₂-D-hArg(Et)₂-Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂;-   Ac-Cys-Lys-Asn-4-Cl-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-D-Cys-NH₂;-   Bmp-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂;-   Bmp-Tyr-D-Trp-Lys-Val-Cys-Phe-NH₂;-   Bmp-Tyr-D-Trp-Lys-Val-Cys-p-Cl-Phe-NH₂;-   Bmp-Tyr-D-Trp-Lys-Val-Cys-β-Nal-NH₂;-   H-D-β-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂;-   H-D-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH₂;-   H-D-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-β-Nal-NH₂;-   H-pentafluoro-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂;-   Ac-D-β-Nal-Cys-pentafluoro-Phe-D-Trp-Lys-Val-Cys-Thr-NH₂;-   H-D-β-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-p-Nal-NH₂;-   H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-β-Nal-NH₂;-   H-D-β-Nal-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH₂;-   H-D-p-Cl-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH₂;-   Ac-D-p-Cl-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH₂;-   H-D-Phe-Cys-β-Nal-D-Trp-Lys-Val-Cys-Thr-NH₂;-   H-D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH₂;-   cyclo(Pro-Phe-D-Trp-N-Me-Lys-Thr-Phe);-   cyclo(Pro-Phe-D-Trp-N-Me-Lys-Thr-Phe);-   cyclo(Pro-Phe-D-Trp-Lys-Thr-N-Me-Phe);-   cyclo(N-Me-Ala-Tyr-D-Trp-Lys-Thr-Phe);-   cyclo(Pro-Tyr-D-Trp-Lys-Thr-Phe);-   cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe);-   cyclo(Pro-Phe-L-Trp-Lys-Thr-Phe);-   cyclo(Pro-Phe-D-Trp(F)-Lys-Thr-Phe);-   cyclo(Pro-Phe-Trp(F)-Lys-Thr-Phe);-   cyclo(Pro-Phe-D-Trp-Lys-Ser-Phe);-   cyclo(Pro-Phe-D-Trp-Lys-Thr-p-Cl-Phe);-   cyclo(D-Ala-N-Me-D-Phe-D-Thr-D-Lys-Trp-D-Phe);-   cyclo(D-Ala-N-Me-D-Phe-D-Val-Lys-D-Trp-D-Phe);-   cyclo(D-Ala-N-Me-D-Phe-D-Thr-Lys-D-Trp-D-Phe);-   cyclo(D-Abu-N-Me-D-Phe-D-Val-Lys-D-Trp-D-Tyr);-   cyclo(Pro-Tyr-D-Trp-t-4-AchxAla-Thr-Phe);-   cyclo(Pro-Phe-D-Trp-t-4-AchxAla-Thr-Phe);-   cyclo(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe);-   cyclo(N-Me-Ala-Tyr-D-Trp-t-4-AchxAla-Thr-Phe);-   cyclo(Pro-Tyr-D-Trp-4-Amphe-Thr-Phe);-   cyclo(Pro-Phe-D-Trp-4-Amphe-Thr-Phe);-   cyclo(N-Me-Ala-Tyr-D-Trp-4-Amphe-Thr-Phe);-   cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);-   cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba-Gaba);-   cyclo(Asn-Phe-D-Trp-Lys-Thr-Phe);-   cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-NH(CH₂)₄CO);-   cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-β-Ala);-   cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-D-Glu)-OH;-   cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe);-   cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe-Gly);-   cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);-   cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gly);-   cyclo(Asn-Phe-Phe-D-Trp(F)-Lys-Thr-Phe-Gaba);-   cyclo(Asn-Phe-Phe-D-Trp(NO₂)-Lys-Thr-Phe-Gaba);-   cyclo(Asn-Phe-Phe-Trp(Br)-Lys-Thr-Phe-Gaba);-   cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Phe(1)-Gaba);-   cyclo(Asn-Phe-Phe-D-Trp-Lys-Thr-Tyr(But)-Gaba);-   cyclo(Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Pro-Cys)-OH;-   cyclo(Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Pro-Cys)-OH;-   cyclo(Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Tpo-Cys)-OH;-   cyclo(Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-MeLeu-Cys)-OH;-   cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe-Phe-Gaba);-   cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe-D-Phe-Gaba);-   cyclo(Phe-Phe-D-Trp(5F)-Lys-Thr-Phe-Phe-Gaba);-   cyclo(Asn-Phe-Phe-D-Trp-Lys(Ac)-Thr-Phe-NH—(CH₂)₃—CO);-   cyclo(Lys-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);-   cyclo(Lys-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);-   cyclo(Orn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba);-   H-Cys-Phe-Phe-D-Trp-Lys-Thr-Phe-Cys-NH₂;    or a pharmaceutically acceptable salt thereof, wherein a disulfide    bond exists in those compounds having two Cys residues and where the    amine nitrogen of each amide peptide bond and the amino group of the    N-terminal amino acid is optionally substituted with a methyl group,    provided that there is at least one said methyl group in the    compound.

In a further aspect the present invention features SRIF agonistscomprising the N-methylated analogs of the SRIF agonists covered byformulae or those specifically recited in the publications set forthbelow.

-   EP Application No. P5 164 EU (Inventor: G. Keri);-   Van Binst, G. et al. Peptide Research 5:8 (1992);-   Horvath, A. et al. Abstract, “Conformations of Somatostatin Analogs    Having Antitumor Activity”, 22nd European peptide Symposium, Sep.    13-19, 1992, Interlaken, Switzerland;-   U.S. Pat. No. 6,001,801 (1999);-   U.S. Pat. No. 4,904,642 (1990);-   U.S. Pat. No. 4,871,717 (1989);-   U.S. Pat. No. 4,853,371 (1989);-   U.S. Pat. No. 4,725,577 (1988);-   U.S. Pat. No. 4,684,620 (1987)-   U.S. Pat. No. 4,650,787 (1987);-   U.S. Pat. No. 4,603,120 (1986);-   U.S. Pat. No. 4,585,755 (1986);-   EP Application 0 203 031 A2 (1986);-   U.S. Pat. No. 4,522,813 (1985);-   U.S. Pat. No. 4,486,415 (1984);-   U.S. Pat. No. 4,485,101 (1984);-   U.S. Pat. No. 4,435,385 (1984);-   U.S. Pat. No. 4,395,403 (1983);-   U.S. Pat. No. 4,369,179 (1983);-   U.S. Pat. No. 4,360,516 (1982);-   U.S. Pat. No. 4,358,439 (1982);-   U.S. Pat. No. 4,328,214 (1982);-   U.S. Pat. No. 4,316,890 (1982);-   U.S. Pat. No. 4,310,518 (1982);-   U.S. Pat. No. 4,291,022 (1981);-   U.S. Pat. No. 4,238,481 (1980);-   U.S. Pat. No. 4,235,886 (1980);-   U.S. Pat. No. 4,224,190 (1980);-   U.S. Pat. No. 4,211,693 (1980);-   U.S. Pat. No. 4,190,648 (1980);-   U.S. Pat. No. 4,146,612 (1979);-   U.S. Pat. No. 4,133,782 (1979);-   U.S. Pat. No. 5,506,339 (1996);-   U.S. Pat. No. 4,261,885 (1981);-   U.S. Pat. No. 4,728,638 (1988);-   U.S. Pat. No. 4,282,143 (1981);-   U.S. Pat. No. 4,215,039 (1980);-   U.S. Pat. No. 4,209,426 (1980);-   U.S. Pat. No. 4,190,575 (1980);-   EP Application 0 363 589 A2 (1990);-   EP Patent No. 0 389 180 (1990);-   EP Application No. 0 505 680 (1982);-   EP Application No. 0 083 305 (1982);-   EP Application No. 0 030 920 (1980);-   PCT Application No. WO 97/01579 (1997);-   PCT Application No. WO 91/18016 (1991);-   PCT Application No. WO 91/09056 (1991);-   PCT Application No. WO 90/12811 (1990);-   PCT Application No. WO 88/05052 (1988);-   U.K. Application No. GB 2,095,261 (1981); and-   French Application No. FR 2,522,655 (1983).

The compounds of formula (I), formula (II) and group (III) of theinstant application are useful for the same uses as SRIF, dependent uponthe binding specificity or lack thereof, as may be determined by thebinding assays described herein.

Thus in another aspect the invention is featured a method of binding oneor more of human somatostatin subtype receptors-1, -2, -3, -4 and -5,which comprises the step of administering one or more compounds offormula (I) and/or formula (II) and/or group (III), or apharmaceutically acceptable salt(s) of such compound or compounds, to arecipient in need thereof.

In a preferred embodiment of the immediately foregoing method isfeatured a method of eliciting a somatostatin agonist effect, whichcomprises the step of administering one or more compounds of formula (I)and/or formula (II) and/or group (III), or a pharmaceutically acceptablesalt(s) of such compound or compounds, to a recipient in need thereof.

In a more preferred embodiment of the immediately foregoing method isfeatured a method of treating a disease or condition in a human or otheranimal in need thereof, which comprises administering one or morecompounds of formula (I) and/or formula (II) and/or group (III), or apharmaceutically acceptable salt(s) of such compound or compounds, tosaid human or other animal, wherein said disease or condition isselected from the group consisting of Cushings Syndrome,gonadotropinoma, hyperparathyroidism, Paget's disease, VIPoma,nesidioblastosis, hyperinsulinism, gastrinoma, Zollinger-EllisonSyndrome, hypersecretory diarrhea related to AIDS and other conditions,irritable bowel syndrome, pancreatitis, Crohn's Disease, systemicsclerosis, thyroid cancer, psoriasis, hypotension, panic attacks,sclerodoma, small bowel obstruction, gastroesophageal reflux,duodenogastric reflux, Graves' Disease, polycystic ovary disease, uppergastrointestinal bleeding, pancreatic pseudocysts, pancreatic ascites,leukemia, meningioma, cancer cachexia, acromegaly, restenosis, hepatoma,lung cancer, melanoma, inhibiting the accelerated growth of a solidtumor, decreasing body weight, treating insulin resistance, Syndrome X,prolonging the survival of pancreatic cells, fibrosis, hyperlipidemia,hyperamylinemia, hyperprolactinemia and prolactinomas.

With the exception of the N-terminal amino acid, all abbreviations(e.g., Phe for A¹) of amino acids in this disclosure stand for thestructure of —NH—CH(R)—CO—, wherein R in the immediately foregoingformula is the side chain of an amino acid (e.g., CH₃ for Ala). For theN-terminal amino acid, the abbreviation stands for the structure of(R¹R²)—N—CH(R)—CO—, wherein R is a side chain of an amino acid and R¹and R² are as defined herein.

The nomenclature for the somatostatin receptor subtypes is in accordancewith the recommendations of IUPHAR, in which SSTR-4 refers to thereceptor originally cloned by Bruno et al., and SSTR-5 refers to thereceptor cloned by O'Carroll et al. Abbreviations of the common aminoacids are in accordance with the recommendations of IUPAC-IUB. Thefollowing are abbreviations of certain α-amino acids as may appearherein:

Abu=α-aminobutyric acid

Aib=α-aminoisobutyric acid;

β-Ala=β-alanine;

Amp=4-amino-phenylalanine;

Ava=5-aminovaleric acid;

Cha=cyclohexylalanine;

Gaba=γ-aminobutyric acid;

Lys=lysine;

β-Nal=β-(2-naphthyl)alanine;

Nle=norleucine;

Nva=norvaline;

Orn=ornithine;

Pal=β-(3-pyridinyl)alanine;

Phe=phenylalanine;

Ser=serine;

hSer=homoserine;

Thr=threonine; and

Tyr=tyrosine.

Additional abbreviations include:

DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;

DCM, dichloromethane;

DIC, dicyclohexylcarbodiimide;

DIEA, diisopropyethylamine;

DMF, dimethylformamide;

MTBD, 1,3,4,6,7,8-Hexahydro-1-methyl-2H-pyrimido[1,2-a]pyrimidine;

NPS, 2-nitrophenylsulfonyl;

TBTU, O-Benzotri-azol-1-yl-N,N,N′,N′-tetramethyluroniumtetrafluoroborate; and

TFA, trifluoroacetic acid.

A compound of the present invention or pharmaceutically acceptable saltthereof can be administered by oral, parenteral (e.g., intramuscular,intraperitoneal, intravenous or subcutaneous injection, or implant),nasal, vaginal, rectal, sublingual or topical routes of administrationand can be formulated with pharmaceutically acceptable carriers toprovide dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than suchinert diluents, e.g., lubricating agents such as magnesium stearate. Inthe case of capsules, tablets and pills, the dosage forms may alsocomprise buffering agents. Tablets and pills can additionally beprepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as coca butter or a suppository wax.

Compositions for nasal or sublingual administration are also preparedwith standard excipients well known in the art.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient be such that a suitable dosage form is obtained. The selecteddosage depends upon the desired therapeutic effect, on the route ofadministration, and on the duration of the treatment. Generally, dosagelevels of between 25 μg/kg/day to 100 mg/kg/day of body weight daily areadministered as a single dose or divided into multiple doses to humansand other animals, e.g., mammals, to obtain the desired therapeuticeffect.

A preferred general dosage range is 250 μg/kg/day to 5.0 mg/kg/day ofbody weight daily which can be administered as a single dose or dividedinto multiple doses.

Further, a compound of the present invention or pharmaceuticallyacceptable salt thereof can be administered in a sustained releasecomposition such as those described in the following patents. Amongthose formulations, 14-day or 28-day slow release formulations will bepreferred. U.S. Pat. No. 5,672,659 teaches sustained releasecompositions comprising a peptide and a polyester. U.S. Pat. No.5,595,760 teaches sustained release compositions comprising a peptide ina gelable form. U.S. Pat. No. 5,821,221 teaches polymeric sustainedrelease compositions comprising a peptide and chitosan. U.S. Pat. No.5,916,883 teaches sustained release compositions comprising a peptideand cyclodextrin. International Patent Application No. PCT/US99/01180,(publication no. WO 99/38536, Aug. 5, 1999), teaches absorbablesustained release compositions of a peptide. The contents of theforegoing patents and applications are incorporated herein by reference.

The use of immediate or of sustained release compositions depends on thetype of indications targeted. If the indication consists of an acute orover-acute disorder, a treatment with an immediate form will bepreferred over the same with a prolonged release composition. On thecontrary, for preventive or long-term treatments, a prolonged releasecomposition will generally be preferred.

DETAILED DESCRIPTION OF THE INVENTION

One skilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent. The following specificembodiments are, therefore, to be construed as merely illustrations ofthe invention and is not meant to be construed as limiting the fullscope of the invention.

Synthesis

4-Methylbenzhydrylamine hydrochloride resin (0.25 or 0.5 mequiv g⁻¹) wasobtained from Advanced ChemTech Inc., Louisville, Ky. N^(α)tert-Butyloxycarbonyl (Boc) protected amino acids were purchased fromBachem Inc., Torrance, Calif., Advanced ChemTech Inc., and SynthetechInc., Albany, Oreg. The reactive side-chains of the amino acids weremasked with one of the following groups: Cys, 4-methylbenzyloxycarbonyl;Lys, 2-chlorobenzyloxycarbonyl; Thr, O-benzyl; Tyr,O-2,6-dichlorobenzyl. All reagents and solvents were ACS grade or betterand were used without further purification.

Compounds of the present invention, e.g., compounds of formula (I) canbe and were synthesized on 4-methylbenzhydrylamine functionalized, 1%cross-linked polystyrene resin (0.25 or 0.5 mequiv g⁻¹), in 0.25 mmolscale on an Advanced ChemTech (model 200) synthesizer, using thefollowing protocol: deblocking, 40% TFA (2 min, 20 min); DCM wash cycle(three washes); neutralization, 10% DIEA (1 min, 5 min); DMF wash cycle;DCM wash cycle (two washes); double coupling; first with 1,3-diisopropylcarbodiimide esters (3 equiv.), 30 min in DCM; DCM wash (three washes);second coupling with preformed TBTU esters (3 equiv.), 90 min in DMF,with a catalytic amount of DIEA; DMF wash (one wash); DCM wash (threewashes). Coupling reactions were monitored qualitatively with theninhydrin test.

N^(∝)-Protection. After deblocking the amino group at the desiredmethylation site, the resin was suspended in DCM (20 mL). To thissuspension, collidine (3 equiv.) and o-nitrobenzenesulfonyl chloride (3equiv.) were added and the mixture was shaken using Advanced ChemTech(model 200) synthesizer for 2 h. Then the resin was subjected to DCMwash (2 washes) and DMF wash (3 washes). Protection was monitoredqualitatively by the ninhydrin test.

N^(∝)-Methylation. The o-nitrobenzenesulfonamide protected resin wassuspended in DMF (20 mL), to which MTBD (3 equiv.) and methyl4-nitrobenzenesulfonate or dimethyl sulfate (for Cys¹¹) were added. Themixture was shaken using Advanced ChemTech (model 200) synthesizer for0.5 h and the resin was subjected to DMF wash (4 washes).

N^(∝)-Me Deprotection. Once the desired residue was methylated, theresin was again suspended in DMF (20 mL). DBU (3 equiv.) and2-mercaptoethanol (3 equiv.) were added to the suspension and themixture was agitated for 0.5 h in Advanced ChemTech (model 200)synthesizer. Then the resin was thoroughly washed with DMF (5 washes).

Peptide Cleavage. The peptides were cleaved from the resin support withsimultaneous side-chain deprotection by acidolysis using anhydroushydrogen fluoride containing the scavenger anisole (˜30% v/v) for 45 minat 0° C. The peptides were cyclized in 90% acetic acid (˜600 mL) with aslight excess of 12 (15 min). Excess 12 was then removed by the additionof ascorbic acid.

Purification. The crude peptides were purified by preparative RP-HPLC onC-18 bonded silica gel using axial compression columns (Dynamax-300 Å, 5or 8 μm, 21.4×250 mm). A linear gradient elution system at a flow rateof 20 mL min⁻¹ was employed: A; 0.1% TFA, B; 0.1% TFA in 80% MeCN, 20% Bto 50% B at 1% min⁻¹. The separations were monitored by analyticalRP-HPLC at 215 nm. The fractions containing the product were pooled,concentrated in vacuo and subjected to lyophilization. Each peptide wasobtained as a fluffy white powder of constant weight by lyophilizationfrom aqueous acetic acid. The purity of the final peptides was assessedat 215 nm by analytical RP-HPLC. Analytical RP-HPLCs were recorded usinga Vydac C-18 support (4.6□250 mm, 5 μm, 300 Å pore size, LiquidSeparations Group). The linear gradient system was used at a flow rateof 1.5 mL min⁻¹: HPLC-1, A, 0.1% TFA; B, 0.1% TFA in 80% MeCN; 20% B to50% B at 1% min⁻¹; HPLC-2, C, 5% MeCN in TEAP (0.1 M, pH 3); D, 20% C inMeCN, 10% D to 70% D at 1% min⁻¹. Column eluent was monitored at 215 nm.The retention time and purity of each peptide was assessed by the RaininDynamax HPLC Method Manager.

Amino Acid Analysis. The peptides were hydrolyzed in vacuo (110° C.; 20h) in 4 M methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole(Pierce). Amino acid analyses were performed on the hydrolyzatesfollowing derivatization with o-phthalidaldehyde reagent (Sigma ChemicalCo.) using an automatic HPLC system (Rainin Instrument Co.) fitted witha 100×4.6 mm, 3 μm C18 axial compression column with integral guardcolumn (Microsorb AAAnalySiS™, Type O; Rainin Instrument Co.) Thederivatized primary amino acids were eluted using a binary gradient ofbuffer A; 0.10 M sodium acetate containing 4.5% v/v methanol and 0.5%v/v tetrahydrofuran at pH 7.2 and buffer B; methanol. The gradientsequence; 0% A at 0 min; 35% A at 16.5 min; 90% A at 30 min and 90% A at33 min was used with a flow rate of 1.0 mL min⁻¹ at ambient temperature.Eluent was monitored at 340 nm and integrated by the Dynamax HPLC MethodManager (Rainin). Standard retention times were as follows: Asp, 6.6min; Arg, 19.9 min; Trp, 25.4 min and Lys, 29.5 min. Each peptide ofTable I produced the expected analytical results for the primary aminoacids. Cysteine was not quantified.

Mass Spectrometry. Peptides were analyzed by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry using a LaserMat2000 mass spectrometer (Thermal Bioanalysis, San Jose, Calif.) usingα-cyano-4-hydroxycinnamic acid as the matrix with Substance P (1348.7Da) as an internal standard. In each case, the spectra consisted of amajor M-H ion peak for the internal standard, the expected analyte M-Hpeak, and a few peaks associated with the matrix (<500 Da). Mass valuesso derived for certain representative compounds of the instant inventionare detailed in Table 1.

SRIF Analogue Inhibition of GH Release. Anterior pituitaries from adultmale rats were collected and dispersed by a previously describedtrypsin/DNase method. (Murphy, W. A.; Taylor, J.; Moreau, J.-P. and Coy,D. H., Peptide Res. 1989, 2, 128-132.) The dispersed cells were dilutedwith sterile-filtered Dulbecco's modified Eagle medium (MEM, GibcoLaboratories, Grand Island, N.Y.), which was supplemented with 2.5%fetal calf serum (Gibco), 3% horse serum (Gibco), 10% fresh rat serum(stored on ice for no longer than 1 h) from the pituitary donors, 1% MEMnonessential amino acids (Gibco), gentamycin (10 ng mL⁻¹; Sigma) andnystatin (10,000 U mL⁻¹; Gibco). The cells were randomly plated at adensity of approximately 200,000 cells/well (Costar cluster 24;Rochester Scientific Co., Rochester, N.Y.). The plated cells weremaintained in the above Dulbecco's medium in a humidified atmosphere of95% air/5% CO₂ at 37° C. for 4-5 days. In preparation for a hormonechallenge, the cells were washed with medium 199 (Gibco, 3×1 mL). Eachdose of a compound of this invention was tested in triplicate wells in atotal volume of 1 mL medium 199 containing 1% BSA (fraction V; SigmaChemical Co.). All wells contained GHRH(1-29)NH₂ (1 nM). Afterincubation in an air/carbon dioxide atmosphere (95/5%, 3 h at 37° C.),the medium was removed and stored at −20° C. until assayed for hormonecontent. Growth hormone in media was measured by a standard doubleantibody RIA using components generously supplied by Dr. A. F. Parlow atthe National Hormone and Pituitary Program (NHHP) Torrance, Calif.Agonist IC₅₀'s were calculated using Sigmaplot (Jandel Scientific, SanRafael, Calif.). Values are expressed as the mean IC₅₀ (nM)±SEM from (n)separate dose-response curves.

Functional Expression of the Cloned Human Somatostatin Receptors. Thegenomic clones containing the human somatostatin receptors (hSSTR-1 tohSSTR-5) (Yamada, Y., et al. al., Proc. Natl. Acad. Sci. USA. 1992, 89,251-255; Yasuda, K., et al., J. Biol. Chem. 1992, 267, 20422-20428;Yamada, Y., et al., Mol. Pharmacol. 1992, 42, 2136-2142; Rohrer, L., etal., Proc. Natl. Acad. Sci. USA. 1993, 90, 4196-4200.) were kindlyprovided by Dr. Graeme I. Bell of the University of Chicago. ThehSSTR-1, hSSTR-2, hSSTR-3, hSSTR-4 and hSSTR-5 cDNAs were isolated as a1.5-kb PstI-XmnI fragment, 1.7-kb BamHI-HindIII fragment, 2.0-kbNcoI-HindIII fragment, 1.4-kb NheI-NdeI fragment, and a 1.2-kbHindIII-XbaI fragment, respectively, each containing the entire codingregion of the full-length receptors. These fragments were independentlysubcloned into the corresponding restriction endonuclease sites in themammalian expression vector pCMV5, downstream from the humancytomegalovirus (CMV) promoter, to produce the expression plasmidspCMV5/hSSTR-1, pCMV5/hSSTR-2, pCMV5/hSSTR-3, pCMV5/hSSTR-4 andpCMV5/hSSTR-5. For transfection into CHO-K1 cells, a plasmid, pRSV-neo(American Type Culture Collection, Rockville, Md.), carrying theneomycin mammalian cell selectable marker was added.

Receptor Expression and Transfection. Transfections were performed bythe calcium phosphate method. CHO-K1 cells were maintained in α-minimumessential medium (α-MEM; Gibco) supplemented with 10% fetal calf serumand transfected with each of the expression plasmids using calciumphosphate precipitation. Clones that had inherited the expressionplasmid were selected in α-MEM supplemented with 500 μg mL⁻¹ ofgeneticin (G418; Gibco). Independent CHO-K1 clones were picked byglass-ring cloning and expanded in culture in the selective media.Membranes were prepared from the isolated clones and hSSTR expressionwas initially assessed for binding with [¹²⁵I]Tyr¹¹-SRIF and[¹²⁵I]MK-678 (for SSTR-2).

Radioligand Binding Assays. Cell membranes of the 5 cells types wereobtained from homogenates (Polytron setting 6, 15 sec) of thecorresponding CHO-K1 cells, in ice-cold Tris-HCl (50 mM) and centrifuged(39000 g, 10 min×2), with an intermediate resuspension in fresh buffer.The final pellets were resuspended in Tris-HCl (10 mM) for assay.Aliquots of the membranes were incubated (30 min at 37° C.) with 0.05 nM[¹²⁵I]Tyr¹¹-SRIF (types 1, 3, 4, 5) or [¹²⁵I]MK-678 (type 2) in 50 nMHEPES (pH 7.4) containing BSA (10 mg mL⁻¹); MgCl₂ (5 mM), Trasylol (200kIU mL⁻¹), bacitracin (0.02 mg mL⁻¹), and phenylmethanesulfonyl fluoride(0.02 mg mL⁻¹). The final assay volume was 0.3 mL and incubations wereterminated by rapid filtration through GF/C filters pre-soaked in 0.3%poly(ethylenimine) using a Brandel rapid filtration module. Each tubeand filter was then washed with aliquots of cold buffer (3×5 mL).

Specific binding is defined as the total radioligand bound minus thatbound in the presence of 1.0 μM SRIF. The following total radioligandbinding and non-specific binding (nsb) values were typically obtainedwith these assay systems: hSSTR-1, 7000 cpm total versus 3500 cpm nsb;hSSTR-2, 9000 cpm total versus 1000 cpm nsb; hSSTR-3, 8000 cpm totalversus 1000 cpm nsb; hSSTR-4, 6000 cpm total versus 3500 cpm nsb; andhSSTR-5, 7500 cpm total versus 3500 cpm nsb. The binding affinities areexpressed as K_(i) values ±SEM (nM) for each of the five receptorsubtypes. Ki values derived for representative compounds of the instantinvention are detailed in Table 2.

Molecular Modeling. All molecular modeling was performed on a SiliconGraphics Indigo² High Impact 10000 computer, using SYBYL 6.6 with theKollman all atom force field. The PDB files for the three solution NMRstructures of the initial compound Sandostatin/OCTREOTIDE®;DPhe⁵-c[Cys⁶-Phe⁷-DTrp⁸-Lys⁹-Thr¹⁰-Cys¹¹]-Thr¹²-ol (1SOC and 2SOC) wereobtained from the PDB database. These structures were imported intoSYBYL6.6 and mutated to form the N-methylated compounds based on Example9. The Kollman partial atomic charges were loaded from the monomerdictionary. The structures were optimized by annealing the mutatedresidue and then by full energy minimization using the conjugategradient algorithm to a final root mean square (rms) gradient of ≦0.01Kcal mol.Å⁻¹. A distance-dependent dielectric function was employedtogether with the default settings for all the other minimizationoptions.

Examples 9 and 18 were alkylated at every residue by a solid phaseprocedure whilst being assembled on methylbenzhydrylamine resin. Afterthe tert-butoxycarbonyl (Boc) group was removed at the desiredN-methylation site, the free amine of the resin bound peptide wasprotected using o-nitrobenzenesulfonyl chloride and collidine indichloromethane. Then the amide N—H of o-nitrobenzenesulfonamide wasselectively deprotonated by the strong, hindered, non-ionic base MTBDand methylated using methyl p-nitrobenzenesulfonate in DMF. Themethylated sulfonamide was deprotected by β-mercaptoethanol and DBU inDMF and this reaction was easily followed by the appearance of brightyellow color in the solution, indicating the removal ofo-nitrobenzenesulfonyl group from the resin bound peptide. Also, thisdeprotection was slower if the N-sulfonamide was not alkylated, thuscapping the unalkylated peptide. The subsequent amino acid was coupledtwo times using TBTU/DIPEA instead of DIC.

The sequence (o-NBS)HN-Cys¹¹(4-MeZ)-Thr¹²(OBzl)-® could not bemethylated using methyl o-nitrobenzenesulfonate. This problem was,however, circumvented by using dimethyl sulfate as a methylating agentinstead of the bulky methyl o-nitrobenzenesulfonate.

The binding affinities (Kd, nM) of all SRIF analogues were determinedusing their concentration-dependent displacement of ¹²⁵I-radiolabeledpeptide ligands from membranes isolated from CHO cells transfected withthe corresponding human somatostatin receptor. For reference, thebinding affinities of SRIF-14 and SRIF-28 in the same system were used.SRIF-28 displays particularly high affinity for type 5 receptorscompared to SRIF-14. Given the profound effect which the conformationand side-chain of the N-terminal amino acid has on the biologicalactivities of this type of analogue, two series of base structure(compounds 9 and 18) were used for the present study—one containing aDPhe (analogue 9) and the other a Tyr residue (analogue 18) to give atotal of 16 N-methylated analogues, the structures and physicochemicalcharacteristics of which are given in Table 1.

The compounds of the present invention were synthesized as describedabove and/or as described in the various references cited herein. TABLE1 N-Methyl Analogue Structures and Analytical Data Mass Spectrum Ex.Sequence (M − H⁺) HPLC^(c) No. A¹-cyclo{Cys-A²-D-Trp-A³-A⁴-Cys}-A⁵-Y¹Calcd.^(a) Obsd.^(b) (t_(R−1))^(d) (t_(R−1))^(e) 1NMeDPhe-cyclo(Cys-Phe-DTrp-Lys-Thr-Cys)-Thr-NH₂ 1047.3 1047.7 12.7 12.62 DPhe-cyclo(NMeCys-Phe-DTrp-Lys-Thr-Cys)-Thr-NH₂ 1047.3 1048.0 16.316.4 3 DPhe-cyclo(Cys-NMePhe-DTrp-Lys-Thr-Cys)-Thr-NH₂ 1047.3 1048.814.4 14.8 4 DPhe-cyclo(Cys-Phe-NMeDTrp-Lys-Thr-Cys)-Thr-NH₂ 1047.31047.3 16.6 17.3 5 DPhe-cyclo(Cys-Phe-DTrp-NMeLys-Thr-Cys)-Thr-NH₂1047.3 1047.6 15.6 15.7 6DPhe-cyclo(Cys-Phe-DTrp-Lys-NMeThr-Cys)-Thr-NH₂ 1047.3 1047.8 9.4 9.4 7DPhe-cyclo(Cys-Phe-DTrp-Lys-Thr-NMeCys)-Thr-NH₂ 1047.3 1047.8 13.2 12.18 DPhe-cyclo(Cys-Phe-DTrp-Lys-Thr-Cys)-NMeThr-NH₂ 1047.3 1048.1 10.910.8 9 DPhe-cyclo(Cys-Phe-DTrp-Lys-Thr-Cys)-Thr-NH₂ 1033.2 1032.7 12.912.3 10 NMeTyr-cyclo(Cys-Phe-DTrp-Lys-Thr-Cys)-Thr-NH₂ 1063.3 1063.813.4 13.7 11 Tyr-cyclo(NMeCys-Phe-DTrp-Lys-Thr-Cys)-Thr-NH₂ 1063.31063.7 14.3 14.7 12 Tyr-cyclo(Cys-NMePhe-DTrp-Lys-Thr-Cys)-Thr-NH₂1063.3 1063.4 13.6 13.4 13Tyr-cyclo(Cys-Phe-NMeDTrp-Lys-Thr-Cys)-Thr-NH₂ 1063.3 1063.7 15.8 15.914 Tyr-cyclo(Cys-Phe-DTrp-NMeLys-Thr-Cys)-Thr-NH₂ 1063.3 1063.2 17.718.2 15 Tyr-cyclo(Cys-Phe-DTrp-Lys-NMeThr-Cys)-Thr-NH₂ 1063.3 1063.011.5 11.7 16 Tyr-cyclo(Cys-Phe-DTrp-Lys-Thr-NMeCys)-Thr-NH₂ 1063.31063.4 14.2 14.2 17 Tyr-cyclo(Cys-Phe-DTrp-Lys-Thr-Cys)-NMeThr-NH₂1063.3 1063.7 11.6 11.9 18 Tyr-cyclo(Cys-Phe-DTrp-Lys-Thr-Cys)-Thr-NH₂1049.2 1050.0 13.6 13.6^(a)Theoretical molecular weight (M − H+, Da).^(b)Observed molecular weight (M − H+, Da).^(c)Reversed-phase HPLC (C-18, 5 μm, 4.6 × 250 mm, λ = 215 nm) retentiontimes (min). Each compound was found to have a purity of >98% by HPLC.^(d)HPLC Elution System: A; 0.1% TFA, B; 0.1% TFA in 80% MeCN, 20% B to50% B at 1% min⁻¹ and 1.5 mL min⁻¹.^(e)HPLC-2 elution system: C, 5% MeCN in TEAP (0.1M, pH 3); D, 20% C inMeCN, 10% D to 70% D at 1% min⁻¹ and 1.5 mL min⁻¹.

TABLE 2 Binding Affinities (K_(d)) of Analogues Shown in Table 1 forCloned Human sst₁₋₅ Receptors and Agonist Activity (IC₅₀) on Culture RatPituitary Cells K_(d) ^(a) ± SEM (nM) Agonist IC₅₀ ± Ex. No. hsst₁ hsst₂hsst₃ hsst₄ hsst₅ SEM (n)^(b) (nM) SRIF-14  2.0 ± 0.35  0.25 ± 0.03  1.2± 0.23  2.0 ± 0.25  1.4 ± 0.29 0.17 ± 0.054  SRIF-28  1.9 ± 0.42  0.31 ±0.06  1.3 ± 0.29 5.4 ± 2.5  0.4 ± 0.05 0.23 ± 0.052  1 316 ± 11   1.03 ±0.26 17.9 ± 2.5 >1,000 4.89 ± 1.4  0.32 ± 0.13 (7) 2 378 ± 119  1.04 ±0.18   13 ± 0.5 >1,000  23.71  0.36 ± 0.19 (4) 3 >1,000  13.17 ± 3.85830 ± 86 >1,000 83.24 ± 25.8  7.29 ± 2.08 (2) 4 1,200  23.5 ± 3.92 11.05± 1.03 >1,000  0.61 ± 0.36 18.7 ± 8.1 (2) 5 867 ± 102  1.84 ± 0.21 67.48 ± 10.02 >1,000  8.41 ± 6.85  0.74 ± 0.14 (4)6 >1,000  >1,000 >1,000 >1,000 >1,000   nd^(c) 7 622 ± 172 56.23 ± 26.4 44.4 ± 8.36 574 28.42 ± 19.3 nd^(c) 8 >1,000  14.84 ± 1.53 124.3 ± 11.7182 313   28.8 ± 8.0 (2) 9 761    0.15 ± 0.08 11.84 ± 0.9  >1,000   8.35 0.16 ± 0.04 (5) 10 811 ± 188  9.74 ± 1.87  3.01 ± 1.05 nd^(c) 27.00 ±14.3 11.3 ± 2.5 (5) 11 862 ± 162  8.96 ± 1.66  2.73 ± 2.43 nd^(c) 114.011.9 ± 4.1 (2) 12 653 ± 245 40.09 ± 3.79  94.20 ± 16.71 nd^(c) 94.99 ±22.0  103 ± 4.0 (2) 13 1,000 120.4 ± 22.2 8.00 ± 0.9 nd^(c) 50.38 ± 28.6nd^(c) 14 956 ± 43  14.25 ± 3.12 51.02 ± 6.93 nd^(c)  629 ± 371  27.4 ±14.1 (2) 15 1,000 61.35 ± 6.95  440 ± 126 1,000 92.79 ± 0.7  nd^(c) 161,255 56.23 ± 26.4 17.00 ± 2.75 321  16.89  41.2 ± 31.9 (2) 17 611 ±3.5  26.17 ± 10.3  535 ± 200 353 71.84 ± 15.5 nd^(c) 18 1,000 10.33 ±3.53 18.19 ± 4.21 nd^(c) 32.95 ± 15.3  1.11 ± 0.07 (2)^(a)Expressed as the mean ± SEM, single values indicate the results ofone binding experiment.^(b)Rat in vitro antagonist IC₅₀ (nM) versus SRIF (1.0 nM), expressed asthe mean ± SEM of (n) separate dose response curves.^(c)Not determined.^(d)Not applicable

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not to limit the scope of theinvention. Other aspects, advantages, and modifications are within theclaims. Also, the contents of each references cited herein isincorporated by reference in its entirety.

1. A compound of the formula (I),A¹-cyclo{Cys-A²-D-Trp-A³-A⁴-Cys}-A⁵-Y¹,  (I) wherein: A¹ is anoptionally substituted D- or L-aromatic α-amino acid or optionallysubstituted D- or L-cyclo(C₃₋₆)alkylalanine; A² is an optionallysubstituted aromatic α-amino acid or optionally substitutedcyclo(C₃₋₆)alkylalanine; A³ is Lys or Orn; A⁴ is β-Hydroxyvaline, Ser,hSer, or Thr; A⁵ is β-Hydroxyvaline, Ser, hSer, or Thr; and Y¹ is OH,NH₂ or NHR¹, where R¹ is (C₁₋₆)alkyl; wherein each said optionallysubstituted aromatic α-amino acid and each said optionally substitutedcyclo(C₃₋₆)alkylalanine is optionally substituted with one or moresubstituents each independently selected from the group consisting ofhalogen, NO₂, OH, CN, (C₁₋₆)alkyl, (C₂₋₆)alkenyl, (C₂₋₆)alkynyl,(C₁₋₆)alkoxy, Bzl, O-Bzl, and NR⁹R¹⁰, where R⁹ and R¹⁰ each isindependently H or (C₁₋₆) alkyl; and wherein the amine nitrogen of eachpeptide bond and the amino group of A¹ of formula (I) is optionallysubstituted with a methyl group, provided that there is at least onesaid methyl group; and further provided that said compound is notD-Phe-cyclo{Cys-Phe-D-Trp-Lys-(N-Me-Thr)-Cys}-Thr-NH₂; or apharmaceutically acceptable salt thereof.
 2. A compound according toclaim 1, wherein: A¹ is Phe, D-Phe, Tyr, D-Tyr, β-Nal, D-β-Nal, Cha orD-Cha; A² is Phe, Tyr, β-Nal or Cha; and Y¹ is OH or NH₂; or apharmaceutically acceptable salt thereof.
 3. A compound according toclaim 2, wherein A¹ is D-Phe; or a pharmaceutically acceptable saltthereof.
 4. A compound according to claim 2, wherein A¹ is Tyr; or apharmaceutically acceptable salt thereof.
 5. A compound according toclaim 2, wherein A² is Phe; or a pharmaceutically acceptable saltthereof.
 6. A compound according to claim 2, wherein A³ is Lys; or apharmaceutically acceptable salt thereof.
 7. A compound according toclaim 2, wherein A⁴ is Thr; or a pharmaceutically acceptable saltthereof.
 8. A compound according to claim 2, wherein A⁵ is Thr; or apharmaceutically acceptable salt thereof.
 9. A compound according toclaim 3, wherein the compound is(N-Me-D-Phe)-{Cys-Phe-D-Trp-Lys-Thr-Cys}-Thr-NH₂.
 10. A compound of theformula (II),

wherein A¹ is a D- or L- isomer of Ala, Leu, Ile, Val, Nle, Thr, Ser,β-Nal, β-Pal, Trp, Phe, 2,4-dichloro-Phe, pentafluoro-Phe, p-X-Phe, oro-X-Phe, wherein X is CH₃, Cl, Br, F, OH, OCH₃ or NO₂; A² is Ala, Leu,Ile, Val, Nle, Phe, β-Nal, pyridyl-Ala, Trp, 2,4-dichloro-Phe,pentafluoro-Phe, o-X-Phe, or p-X-Phe, wherein X is CH₃, Cl, Br, F, OH,OCH₃ or NO₂; A³ is pyridyl-Ala, Trp, Phe, β-Nal, 2,4-dichloro-Phe,pentafluoro-Phe, o-X-Phe, or p-X-Phe, wherein X is CH₃, Cl, Br, F, OH,OCH₃ or NO₂; A⁶ is Val, Ala, Leu, Ile, Nle, Thr, Abu, or Ser; A⁷ is Ala,Leu, Ile, Val, Nle, Phe, β-Nal, pyridyl-Ala, Trp, 2,4-dichloro-Phe,pentafluoro-Phe, o-X-Phe, or p-X-Phe, wherein X is CH₃, Cl, Br, F, OH,OCH₃ or NO₂; A⁸ is a D- or L-isomer of Ala, Leu, Ile, Val, Nle, Thr,Ser, Phe, β-Nal, pyridyl-Ala, Trp, 2,4-dichloro-Phe, pentafluoro-Phe,p-X-Phe, or o-X-Phe, wherein X is CH₃, Cl, Br, F, OH, OCH₃ or NO₂; eachR₁ and R₂, independently, is H, lower acyl or lower alkyl; and R₃ is OHor NH₂; provided that at least one of A¹ and A⁸ and one of A² and A⁷must be an aromatic amino acid; and further provided that A¹, A², A7 andA⁸ cannot all be aromatic amino acids; wherein the amine nitrogen ofeach of the amide peptide bond is optionally substituted with a methylgroup provided that there is at least one said methyl group in acompound of formula (II).
 11. A compound according to claim 10 whereinsaid compound is selected from the group consisting of:H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Thr-Phe-Thr-NH₂;H-D-Phe-p-NO₂-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH₂;H-D-Nal-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH₂;H-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH₂;H-D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH₂;H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH₂; andH-D-Phe-Ala-Tyr-D-Trp-Lys-Val-Ala-β-D-Nal-NH₂; wherein the amino groupof each of the amide peptide bonds and the N-terminal amino acid isoptionally substituted with a methyl group, provided that there is atleast one such methyl group in the compound.
 12. A compound of theformula

wherein the amine nitrogen of each of the amide peptide bonds and theamino group of the sulfonamide bond is optionally substituted with amethyl group provided that there is at least one such methyl group inthe compound.
 13. A method of eliciting a somatostatin agonist effect,which comprises the step of administering a compound of claim 10 or apharmaceutically acceptable salt thereof to a recipient in need thereof.14. A method of treating a disease or condition in a human or otheranimal in need thereof, which comprises administering a compound ofclaim 10 or a pharmaceutically acceptable salt thereof to said mammal,wherein said disease or condition is selected from the group consistingof Cushings Syndrome, gonadotropinoma, hyperparathyroidism, Paget'sdisease, VIPoma, nesidioblastosis, hyperinsulinism, gastrinoma,Zollinger-Ellison Syndrome, hypersecretory diarrhea related to AIDS andother conditions, irritable bowel syndrome, pancreatitis, Crohn'sDisease, systemic sclerosis, thyroid cancer, psoriasis, hypotension,panic attacks, sclerodoma, small bowel obstruction, gastroesophagealreflux, duodenogastric reflux, Graves' Disease, polycystic ovarydisease, upper gastrointestinal bleeding, pancreatic pseudocysts,pancreatic ascites, leukemia, meningioma, cancer cachexia, acromegaly,restenosis, hepatoma, lung cancer, melanoma, inhibiting the acceleratedgrowth of a solid tumor, decreasing body weight, treating insulinresistance, Syndrome X, prolonging the survival of pancreatic cells,fibrosis, hyperlipidemia, hyperamylinemia, hyperprolactinemia andprolactinomas.